1. Field of Invention
The invention relates generally to a method for the detection of malignant cells, and the use of the method to monitor disease progression and response to treatment in cancer patients. In particular, the invention relates to the identification of malignant lymphocytes by PCR amplification of immunoglobulin or T cell receptor genes which are uniquely rearranged in the malignant clone.
2. Description of the Related Art
Lymphoid malignancies are characterized by the proliferation of cells which carry a unique genetic marker by virtue of their rearranged receptor genes, the immunoglobulin (Ig) genes in B cells, and the T cell receptor (TCR) genes in T cells. It is well known in the art that germ line genes for both Ig and TCR exist as pools of gene segments which become assembled during the normal differentiation of B and T lymphocytes by a process of site specific recombination (Alberts et. al., 1995). Diversity of Ig and TCR are generated by the combinatorial association of these gene segments from the different pools, so that the total repertoire of antigen receptors is log-folds greater than the actual number of receptor gene segments.
The Ig genes comprise 3 clusters of genes, on three different chromosomes: (1) the heavy chain (IgH) genes, which include the immunoglobulin heavy chains, (2) k light chain genes and (3) l light chain genes, which encode the immunoglobulin light chains. The IgH cluster consists of four pools of gene segments, known as C (constant), J (joining), D (diversity) and V (variable). There are nine C gene segments, arranged in an ordered cluster, which determine the class of the heavy chain. The variable region of the IgH is encoded by pools comprising 6 J segments, 10 or more D segments and at least 50 V segments. The arrangement of these gene segments on the chromosome is depicted in
FIG. 1. The Ig light chain genes consist of C, J, and V, but no D segment. Similarly, the genes for the a and b and the g and d chains of the TCR exist on separate chromosomes as pools of C, J, D, and V gene segments.
During differentiation of a T or B cell, the germ line genes are rearranged so that one member of each pool of variable region gene segments (J, D, and V) is randomly selected and the selected segments are joined together. The process of site specific recombination that takes place during lymphocyte differentiation is distinctive, in that during the joining of the Ig and TCR gene segments J, D and V, a variable number of nucleotides may be lost from the ends of the recombining gene segments. In the case of IgH recombination only, one or more nucleotides may also be randomly inserted at the joining site. This loss and gain of nucleotides at the joining sites Ig or TCR is a source of further diversity. It yields rearranged Ig or TCR genes which may be different in length. If short regions spanning the junctions of gene segments (VD, DJ, and JC) are examined, they may be substantially different in length.
The variable regions of both Ig and TCR comprise three regions with little sequence homology between different clones, which are known as xe2x80x9chypervariablexe2x80x9d or xe2x80x9ccomplementarity-determining regionsxe2x80x9d (known as CDR1, CDR2, and CDR3, shown in FIG. 1). The intervening portions of the variable region are more consistent between different clones, and are known as xe2x80x9cframeworkxe2x80x9d (FR1, FR2, FR3, and FR4). The CDR3 region, which is encoded by the VJ junctional region of the light chain, and the D region plus the VD and DJ junctional regions of the heavy chain, is the most highly variable, due to the somatic mutations introduced during recombination, as described above.
Each B lymphocyte expresses only a single rearranged IgH gene, and a single rearranged k or l gene. Each mature T lymphocyte expresses a single TCR a chain and a single TCR b chain. In a lymphoid malignancy, if clonal expansion of a tumor progenitor cell took place after rearrangement of the Ig or TCR gene, it is possible to identify a signature or clonotypic rearrangement which is characteristic of the malignant clone. With the appropriate molecular probes, cells related to the malignant clone can be distinguished from cells with unrearranged Ig or TCR genes, or cells which carry different rearrangements. The rearrangement of Ig or TCR genes in a clone is called its xe2x80x9cclonotypic rearrangementxe2x80x9d.
It is clinically important to be able to detect and characterize tumor cells, not only at diagnosis, but during and after treatment. It is also important to be able to detect any malignant cells that might contaminate a population of stem cells destined for autologous transplantation after ablative chemotherapy. The following methods which are currently available to detect malignant cells carrying a monoclonal or clonotypic rearrangement in patient samples differ in their accuracy and their sensitivity. None are quantitative.
Morphological examination of cells in patient blood samples or biopsies is currently used, but is relatively insensitive in detecting minimal residual disease. Also, cells which might be related to the malignant clone, but are at a different stage of maturation, and thus have a different morphology from the bulk of the tumor cells, are probably missed.
Southern blot hybridization analysis of isolated DNA, a technique which is well known in the art, requires that between 1 and 5 percent of the cells in the patient sample carry a clonotypic rearrangement for it to be detected. Although this technique has been used to detect a monoclonal population of cells which are present in high frequency, it is not useful for the detection of minimal residual disease, because it is not sensitive enough. Also, it cannot provide sequence information that definitively characterizes a malignant clone.
Recently, techniques have been developed which rely upon the use of the polymerase chain reaction (PCR) to amplify clonotypic DNA rearrangements in malignant cells. PCR, which is well known in the art (U.S. Pat. No. 4,683,202 to Mullis, 1987), is a process of repeated cycles of DNA denaturation, followed by DNA synthesis which is used to amplify segments of DNA between two fixed anchor points on a DNA molecule.
Single stranded oligonucleotide primers, called PCR primers, are constructed (based on previously obtained nucleic acid sequence information) which will hybridize to the anchor points, one primer, the upstream primer, on the sense strand, and the other primer, the downstream primer, on the antisense strand. The DNA segment is heat denatured, and then cooled to a temperature at which the PCR primers will anneal to their complementary sequences on the DNA segment. A heat-stable DNA polymerase enzyme then copies the DNA between the two anchor points. In 20-40 or more successive cycles of denaturation and DNA synthesis, the DNA segment of interest (between the two anchor points) can be amplified a million-fold or more. The two anchor points must be within a few thousand nucleotides of each other for efficient amplification to occur. In RT-PCR (reverse transcriptase PCR), the RNA in cells is used as the template. It is first copied into cDNA using the enzyme reverse transcriptase, and the resultant cDNA is subjected to the PCR reaction.
In the context of clonotypic rearrangements, xe2x80x9cconsensusxe2x80x9d or xe2x80x9cframeworkxe2x80x9d PCR primers which hybridize to DNA in the constant or framework regions of rearranged Ig or TCR are used to amplify DNA prepared from patient blood or bone marrow samples containing a high proportion of tumor cells. For example, the upstream primer might be chosen from the 5xe2x80x2 end of the V segment, and the downstream primer from the J segment. Germ line (unrearranged) DNA will not be amplified to detectable levels because the distance between the primers is too great for efficient synthesis. A monoclonal rearrangement can often (but not always) be detected as a single band if the amplified DNA is electrophoresed on an appropriate gel. This amplified DNA represents the putative hypervariable region containing the clonotypic V(D)J rearrangement. More specific PCR primers, referred to as patient-specific PCR primers can be designed once the clonotypic sequence has been determined. The following prior art utilizes PCR technology.
U.S. Pat. No. 5,418,132 to Morley (1995) teaches a method for the diagnosis of leukemia and lymphoma by PCR amplification of Ig or TCR gene segments using consensus framework primers, followed by separation of the PCR products on the basis of size. Because rearranged Ig or TCR genes vary somewhat in their size, as noted above, a clonotypic rearrangement that has been amplified can often be detectable as a discrete band on a gel. If the patient sample does not contain a monoclonal population of cells, size separation will yield a smear, with no detectable discrete band. However, the inventors disclose that this method fails to produce a discrete band with every patient sample, even when multiple pairs of primers are employed. This method could be useful during initial diagnosis when the tumor burden is high, but is not proposed as a means of following minimal residual disease after treatment, because it does not involve detection of a specific clonotypic rearrangement.
Flow cytometry-based fluorescent in situ hybridization (FISH) using immunoglobulin heavy chain variable region probes (Cao et al., 1995a; 1995b) has been suggested as a method to detect clonotypic rearrangements in individual cells in myeloma. The FISH technique involves hybridization of a biotin-labeled anti-sense RNA probe to the unamplified RNA in fixed cells in suspension. Cells which contain RNA complementary to the clonotypic sequence are then detected by means of flow cytometry. The sequence of the anti-sense RNA probes are derived by amplifying the mRNA expressed in myeloma patients"" bone marrow mononuclear cells using RT-PCR from homogenized total RNA with consensus framework PCR primers for the IgH variable region. Because the RNA in the cells to be assayed is not amplified, this method is only sensitive enough to detect cells in which the clonotypic RNA is highly abundant, but it cannot detect cells with a low level of the RNA, or cells in which the DNA is rearranged, but is not being transcribed. For example, FISH might work well with myeloma plasma cells, which are virtual xe2x80x9cimmunoglobulin factoriesxe2x80x9d, and contain extremely high concentrations of mRNA encoding the Ig being produced by the cell. However, FISH would not be sensitive enough to detect pre-B cells or B cells which share the clonotypic rearrangement of a myeloma malignant clone, but do not transcribe or have a low level transcription of the gene.
RT-PCR or PCR using patient-specific PCR probes have been used to amplify bulk RNA or DNA preparations made from myeloma patient samples in an attempt to follow minimal residual disease (Billadeau et. al 1991; Billadeau et al, 1992; Billadeau et al., 1993; Chen and Epstein 1996; Cao et al. 1995). In all of these reports, the sequences of the PCR primers were originally derived from amplification of bulk RNA or DNA isolated from tumor-containing material, which the present inventors find may amplify a sequence which is unrelated to the malignant clone. Another major problem with this approach is that the use of bulk nucleic acid to detect clonotypic sequence does not give quantitative results, and may grossly underestimate the frequency of clonotypic sequences.
A reliable method to quantitate malignant cells during assessment of minimal residual disease does not currently exist. A reference by Yameda et al., 1990 proposed a method based on cloning the products of a PCR amplification into bacteriophage and attempting an analysis of the ratio of phage carrying the clonotypic sequence to phage carrying any other rearrangement. Billadeau et al. (1991) proposed to quantitate a PCR amplification of bulk nucleic acid by preparing a standard curve by seeding known numbers of clonotypic cells into a population of non-rearranged cells. Both proposed methods are very indirect. The FISH method of Cao, cited above, while it provides an analysis on a single cell level, is too insensitive to be quantitative, measuring only the cells which express very high levels of mRNA.
In situ PCR and in situ RT-PCR are known in the art as means to detect nucleic acid sequences in single cells (U.S. Pat. No. 5,436,144 to Stewart and Timm, 1995; Nuovo, 1994).
It is clinically important to be able to monitor the members of a malignant T or B lymphocyte clone, over time in order to determine the effect of treatment on cells of malignant lymphocyte clones. Since most of these malignancies are heterogeneous in differentiation state and/or morphology, the only marker that unequivocally confirms a relationship with the malignancy is the immunoglobulin rearrangement of the IgH, light chain (k or l) or the T cell receptor a, b, g or d. Assays using bulk nucleic acid from lymphocyte populations are not quantitative and do not identify the clonotypic cell types present in blood, lymphoid tissue or bone marrow. Often the malignant lymphocyte comprising the major cellular mass of primary tumor divide slowly or not at all, and may be terminally differentiated with little generative capacity. Hematopoietic malignancies appear to be hierarchical with components at sequential states of differentiation not easily detected with conventional clinical assays, especially if they are present at low frequency. Thus at present, the effects of treatment on the full hierarchy of malignant cells in diseases much as myeloma, lymphoma and chronic and acute lymphocytic leukemias, cannot be assessed.